TxA2 production by human arteries and veins

Human arterial and venous segments from patients under-going operations when incubated in Tris buffer both alone and with arachidonic acid were able to produce thromboxane B2 (assessed by radioimmunoassay). Thromboxane B2 (TxB2) production was progressive in time (till 40 min.) and was enhanced by the addition of 1mM norepinephrine. Contamination of tissues by platelet was checked and platelets did not contribute to thromboxane formation. The investigation of the conversion of 1-14C arachidonic acid by vascular tissue indicated that human vascular tissues produce the metabolites of the cyclooxygenase dependent pathway and that prostacyclin is the main metabolite with a PGI2/TxA2 ratio of 4:1. The arterial wall was found to possess an active lipoxygenase dependent pathway. Thromboxane production by intimal cells was negligible and the main source of thromboxane was the media. The production of thromboxane did not change in relation to age, but arterial segments from men produced significantly larger amounts of thromboxane than those from women.     

Metabolism of 7,10,13,16-docosatetraenoic to dihomo-thromboxane 14-hydroxy-7,10,12-nonadecatrienoic acid hydroxy fatty acids by human platelets

Human platelets metabolize 7,10,13,16-docosatetraenoic acid (22:4(n−6) into dihomo-thromboxane B₂ and 14-hydroxy-7,10,12-nonadecatrienoic acid at about twenty percent of the rate they convert arachidonic to thromboxane B₂ and 12-hydroxy-5,8,10-heptadecatrienoic acid. 14-Hydroxy-7,10,12,16-docasatetraenoic was the major metabolite produce via the lipoxygenase pathway. Several other hydroxy were also produced in small amounts via an indomethacin-insensitive pathway. Incubation of 20 μM arachidonic acid with various levels of 22:4(n−6) resulted In a dose-dependent inhibition of both thromboxane B₂ and 12-hydroxy-5,8,10-heptadecatrienoic acid production. Coversely, 12-hydroxy-5,8,10,14-eicosatetraenoic acid synthesis was stimulated because of substrate shunting to the lipoxygenase pathway. These results show that 22:4(n−6) may modify platelet function both by serving as a precursor for a 22-carbon thromboxane and by suppressing the synthesis of thromboxane A₂ from arachidonic acid. In addition, our results suggest that simultaneous release of 22:4(n−6) and arachidonic acid from platelet phospholipids will result in an elevation of both 12-hydroxy-5,8,10,14-eicosatetraenoic acid levels as well as simultaneous synthesis of 14-hydroxy-7,10,12,16-docosatetraenoic acid.     

Inhibitory action of soluble elastin on thromboxane B2 formation in blood platelets

Soluble elastin, prepared from insoluble elastin by treatment with oxalic acid or elastase, was found to inhibit the formation of thromboxane B₂ both from [1-¹⁴C]arachidonic acid added to washed platelets and from [1-¹⁴C]arachidonic acid in prelabeled platelets on stimulation with thrombin. In both systems, the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was accelerated. Oxalic acid-treated soluble elastin st 1 and 10 mg/ml inhibited the formation of thromboxane B₂ from exogenously supplied arachidonic acid 21 and 59%, respectively, and the formation of thromboxane B₂ in prelabeled platelets stimulated by thrombin 44 and 94%, respectively. These concentrations of elastin increased the formation of 12-HETE from exogenously supplied arachidonic acid about 3.4- and 7.3-times, respectively. Almost all the added arachidonic acid was converted to metabolites. In prelabeled platelets, soluble elastin at 1 and 10 mg/ml increased the formation of 12-HETE stimulated by thrombin about 1.3- and 2.8-times, respectively, and inhibited the thrombin-induced total productions of thromboxane B₂ (12-hydroxy-5,8,10-heptadecatrienoic acid (12-HETE) and free arachidonic acid by 26 and 25%, respectively. Elastase-treated digested elastin also inhibited the formation of thromboxane B₂ and stimulated the formation of 12-HETE in prelabeled platelets stimulated by thrombin. This inhibitory action of elastin was not replaced by desmosine. The level of cAMP in platelets was not affected by soluble elastin. Soluble elastin was also found to inhibit platelet aggregation induced by thrombin. However, the inhibitory action of soluble elastin on platelet aggregation cannot be explained by inhibition of thromboxane B₂ formation by the elastin.     

A comparative study of thromboxane and prostacyclin release from ex vivo cultured bovine vascular endothelium

Thromboxane B₂, 6-keto-Prostaglandin F_1α, and Prostaglandin E₂ release have been quantitated from cultured adult by bovine endothelial cell monolayers and from $\text{ex Vivo}$ vascular segments employing specific radioimmunoassay and thin layer chromatography. Release of all three prostaglandins was demonstrable from both endothelial cell systems under basal conditions and following exposure to the ionophore A23187 and arachidonic acid. In culture, the quantity of 6-keto-PGF_1α released was diminished compared to amounts released from the vessel segments while thromboxane B₂ and prostaglandin E₂ release were similar in the two endothelial model systems. However, the amount of thromboxane B₂ assayed was small and the quantity of thromboxane A₂ it represents is probably of little $\text{in Vivo}$ significance to prostacyclin.     

Selective inhibition of prostaglandin endoperoxide thromboxane isomerase by 1-carboxyalkylimidazoles

1-Carboxyalkylimidazoles inhibited the conversion of prostaglandin H₂ to thromboxane B₂ and 12L-hydroxy-5, 8, 10- heptadecatrienoic acid by a partially purified enzyme (prostaglandin endoperoxide thromboxane isomerase) from bovine platelet microsomes. The degree of the inhibition was dependent on the length of carboxyalkyl chain. 1-Carboxyheptylimidazole was the most potent inhibitor, and an almost complete inhibition was obtained at a concentration on the order of 1 μM. The inhibition, as examined with 1-carboxyheptylimidazole, was of noncompetitive type. These 1-carboxyalkylimidazoles did not affect the formation of prostaglandin H₂ from arachidonic acid. Such a selective inhibition was also demonstrated by the reaction of bovine platelet microsomes with arachidonic acid in the presence of 1-carboxyheptylimidazole, resulting in the accumulation of prostaglandin H₂ as an intermediate. Furthermore, a series of 1-alkylimidazoles with no carboxyl group also inhibited the isomerase at higher concentrations. However, the inhibition was not specific for the isomerase; namely, the prostaglandin H₂ formation from arachidonic acid was also affected.     

Prostaglandin E2, prostaglandin E1 and thromboxane B2 release from human monocytes treated with C3b or bacterial lipopolysaccharide

C3b or lipopolysaccharide treatment of human peripheral blood monocytes in culture stimulates an early release of thromboxane B₂ and a delayed release of prostaglandin E into culture supernatants. Immunoreactive thromboxane B₂ release is maximal from 2–8 h, whereas prostaglandin E release is maximal from 16–24 h after stimulation of monocytes in culture. We further examined this process by comparing the time course of labelled prostaglandin E₂, prostaglandin E₁ and thromboxane B₂ release from human monocytes which were pulse or continuously labelled with [³H]arachidonic acid and [¹⁴C]eicosatrienoic acid. The release of labelled eicosanoids was compared with the release of immunoreactive prostaglandin E and thromboxane B₂. The time course of prostaglandin E₂ release was virtually identical to the release of prostaglandin E₁ in all culture supernatants regardless of labelling conditions. However, release of immunoreactive prostaglandin E paralleled the release of labelled prostaglandin E₁ and E₂ only for continuously labelled cultures. The release of labelled prostaglandin E₁ and E₂ from pulse labelled cultures paralleled the release of thromboxane B₂ and not immunoreactive prostaglandin. In contrast, labelled and immunoreactive thromboxane B₂, quantitated in the same culture supernatants, demonstrated similar release patterns regardless of labelling conditions. These findings indicate that the differential pattern of prostaglandin E and thromboxane B₂ release from human monocytes is not related to a time-dependent shift in the release of prostaglandin E₁ relative to prostaglandin E₂. Because thromboxane B₂ and prostaglandin E₂ are produced through cyclooxygenase mediated conversion of arachidonic acid, these results further suggest that prostaglandin E₂ and thromboxane B₂ are independently metabolized in human monocyte populations.     

Stimulation of human T-lymphocyte chemokinesis by arachidonic acid.

The migration of human T lymphocytes, assessed in modified Boyden chambers, was chemokinetically stimulated by arachidonic acid in a dose-related manner that achieved a peak level of 127 ± 34% enhancement (mean ± SD) at 8 μM arachidonic acid. The chemokinetic effect was dependent on the metabolism of the arachidonic acid by the T lymphocytes as derivatives of arachidonic acid that do not serve as prostaglandin and thromboxane precursors were without effect, while the cyclo-oxygenase inhibitors indomethacin (ID₅₀ = 10 μM) and 5,8,11,14-eicosatetraynoic acid (ETYA) (ID₅₀ = 20 μM) suppressed the stimulation of migration by arachidonic acid. The cyclo-oxygenase product 12-l-hydroxy-5,8,10-heptadecatrienoic acid (HHT) reproduced part of the chemokinetic effect of arachidonic acid, but the lipoxygenase product 12-l-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) as well as PGE₂, PGF_2α, and thromboxane B₂ had no stimulatory activity. The ability of ETYA, but not indomethacin, to suppress the migration of unstimulated T lymphocytes suggested that a lipoxygenase metabolite of endogenous arachidonic acid contributes to the maintenance of their normal levels of spontaneous migration.     

Effect of prostaglandins and their precursors on the proliferation of human lymphocytes and their secretion of tumor necrosis factor and various interleukins

Cytokines, released by T cells, participate in inflammation and produce tissue injury. Excess production of cytokines such as interleukins (ILs) and tumor necrosis factor (TNF) is believed to be involved in the pathobiology of conditions such as septicemia and septic shock, collagen vascular diseases, glomerulonephritis etc. On the other hand, prostaglandins (PGs) are known to modulate inflammation, immune response, and T-cell response to antigens. But relatively little information is available on the effects of PGs and PG precursors on the release of cytokines. Here the authors present data which suggests that PGs including thromboxane B₂ (TXB₂) and their precursors such as dihomo-gamma linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid (EPA) can inhibit T-cell proliferation and influence their ability to secrete IL-2, IL-4, IL-6 and TNF in vitro. These results may have relevance to the use of PG-precursors in various inflammatory conditions including collagen vascular diseases.     

Thromboxane-induced pulmonary vasoconstriction: involvement of calcium

Infusion of tert-butyl hydroperoxide (t-bu-OOH) or arachidonic acid into rabbit pulmonary arteries stimulated thromboxane B2 (TxB2) production and caused pulmonary vasoconstriction. Both phenomena were blocked by cyclooxygenase inhibitors or a thromboxane synthase inhibitor. The increase in pulmonary arterial pressure caused by either t-bu-OOH or arachidonic acid infusion correlated with the concentration of TxB2 in the effluent perfusate. The concentration of TxB2 in the effluent perfusate, however, was always 10-fold greater after arachidonic acid infusion. In the rabbit pulmonary vascular bed lipoxygenase products did not appear involved in the vasoactive response to t-bu-OOH or exogenous arachidonic acid infusion. Calcium entry blockers or a calcium-free perfusate prevented the thromboxane-induced pulmonary vasoconstriction. Calmodulin inhibitors also blocked the pulmonary vasoconstriction induced by t-bu-OOH without affecting the production of TxB2 or prostacyclin. These results suggest that thromboxane causes pulmonary vasoconstriction by increasing cytosol calcium concentration.     

Endogenous formation of the prostaglandin endoperoxine metabolite,thromboxane B2, by brain tissue

Thromboxane B₂ was formed from endogenous precursors during short incubations of guinea pig and rat cerebral cortex. The amount formed by guinea pig brain tissue was 5–6 times the formation of prostaglandin F_2α and E₂. Noradrenalin stimulated and indomethacin and mercaptoethanol inhibited thromboxane B₂ formation. The mass spectrum of the brain compound was identical to thromboxane B₂ formed from arachidonic acid by guinea pig lung and human platelets.     

Kinetic studies on the conversion of prostaglandin endoperoxide PGH2 by thromboxane synthase

We have investigated the time course of formation of thromboxane A₂, thromboxane B₂, and the C-17 hydroxy fatty acid, HHT, from arachidonic acid in a washed human platelet suspension. Our results indicate that HHT is not a breakdown product of thromboxane A₂, but rather thromboxane A₂ decomposes exclusively into thromboxane B₂. The kinetics of formation of thromboxane B₂ from the endoperoxide prostaglandin H₂ in human platelet microsomes was examined. Our data suggest that a bimolecular reaction is involved in the formation of thromboxane A₂ from prostaglandin H₂ and that thromboxane synthase is not an isomerase, but may be acting via a dismutase-type reaction. One possibility is that thromboxane and HHT are produced simultaneously from two molecules of prostaglandin H₂.     

Prostanoid biosynthesis in patients with cystic fibrosis

The urinary excretion rate (ng/h/1.73 m²) of prostanoids was determined with a capillary gas-liquid chromatographic mass spectrometric method in 19 patients with cystic fibrosis (CF) aged 1–29 years. Patients with CF showed an increased excretion of prostaglandin E₂ metabolites (PGE-M) and thromboxane B₂ and its metabolites at all ages. An imbalance in the excretion pattern of thromboxane B₂ metabolites also suggested a relative impairment of β-oxidation. There was no increased excretion of dinor-6-keto-PGF_1α, indicating normal prostacyclin biosynthesis. No correlation was found to genotype, clinical score, lung function or bacterial colonization but a significant negative relation was found between the main prostanoids in the urine and serum phospholipid levels of essential fatty acids. The results show that, contrary to the generally accepted decrease of prostanoid excretion in essential fatty acid deficiency, patients with CF increase their production parallel to the development of the deficiency. Since prostanoid synthesis is rate limited by arachidonic acid release, our data support a previously presented hypothesis about a pathological regulation of the release of arachidonic acid in CF.     

Effect of various prostaglandins on the release of arachidonic acid from cultured fibroblasts

Effect of various prostaglandins on the release of arachidonic acid from [¹⁴C]arachidonic acid labeled fibroblasts was studied. Prostaglandin(PG) F_2α was found to enhance the release of radioactive arachidonic acid from the cells. The stimulatory effect was dose dependent, and was greater than that of bradykinin. The active compounds can be ranked in potency for the release of arachidonic acid from the pre-labeled cells per cent of control: PGF_2α(200.1%)>PGF_1α (141.8%)>PGD₂ (137.1%)>thromboxane B₂ (113.7%)>PGE₂ (109.4%). On the other hand, PGI₂ showed a strong inhibitory effect on the arachidonic acid release from the pre-labeled cells (the value was only 69% of the control), while 6-ketoPGF_1α, an end metabolite of PGI₂, had no effect.     

Metabolism of thromboxane B2 in the cynomolgus monkey

[5,6,8,9,11,12,14,15-³H₈]-Thromboxane B₂ was injected into the saphenous vein of female cynomolgus monkeys, and blood samples were withdrawn from the contralateral saphenous vein. The compound was eliminated from the circulation with a half-life of about 10 min after an initial rapid disappearnace. Some more polar products appeared with time, and also small amounts of material less polar than thromboxane B₂; however, the dominating compound in all blood samples was unconverted thromboxane B₂.About 45% of the given dose of tritium was excreted into urine in 48 hrs. Several metabolites of thromboxane B₂ were found. The major urinary metabolites was identified as dinorthromboxane B₂ (about 32% of urinary radioactivity). Unconverted thromboxane B₂ was also found in considerable amounts (13% of urinary radioactivity).It is concluded that 1) dehydrogenation at C-12 is not a major pathway in the degradation of this compound, in contrast to metabolism at the corresponding C-15 alcohol group of prostaglandins; 2) after having gained access to the circulation, thromboxane B₂ is the main circulating compound; however, assay of thromboxane B₂ in plasma will be complicated or precluded by large artifactual production of the compound by platelets during sample collection.     

Differential effects of aspirin and dexamethasone on phospolipase A2 and C activities and arachidonic acid release from endothelial cells in respose to bradykinin and leukotriene D4

The CPAE bovine endothelial cell line may be stimulated to produce eicosanoids. Leukotriene D₄ increased the release of arachidonic acid primarily by activating phospholipase A₂ while bradykinin activated the phospholipase C pathway. Cells pretreated with dexamethasone, a phospholipase A₂ inhibitor, no longer responded to stimulation by LTD₄ but did release arachidonic acid when treated with bradykinin. Aspirin blocked bradykinin-stimulated production of arachidonic acid but left the response to LTD₄ unaffected. We conclude that these cells produce eicosanoids by activation of both PLA₂ and PLC, and that the two different methods of arachidonic acid release can be distinguished by using the common anti-inflammatory drugs aspirin and dexamethasone.     

The ability of vascular tissue to produce prostacyclin decreases with age

The ability of aortae from young and mature swine to produce prostacyclin (PGI₂) has been determined. PGI₂ was measured as its hydration product, 6-keto-PGF_1α and assayed by stable isotope dilution GC-MS. There was no significant difference in 6-keto-PGF_1α production between intimal strips from young and mature aortae in the basal state. In the presence of saturating concentrations of arachidonic acid, however, intimal strips from young aortae synthesized twice as much 6-keto-PGF_1α as did older tissues. Fatty acid compositions of young and mature aortae were virtually identical, making dietary differences an unlikely explanation for the age-related decrease in PGI₂ synthesis. Both young and mature vascular tissues produced essentially only PGI₂; insignificant amounts of PGE₂ and PGF_2α were found.     

Inhibition of platelet thromboxane synthetase by L-1-tosylamido-2-phenylethyl chloromethyl ketone

L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) was found to inhibit several aspects of arachidonic acid (20:4) metabolism in human platelets; the primary effect being inhibition of thromboxane synthetase. Thromboxane B₂ (TxB₂) formation from exogenous 20:4 or PGH₂, or from endogenous 20:4, was inhibited by TPCK at concentrations between 0.1 and 0.5 mM. Formation of malondialdehyde (MDA) and 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT), products which also arise from PGH₂, was inhibited to a similar extent. Inhibition of formation from 20:4 of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), the product of the lipoxygenase pathway, was observed; although the extent of this inhibition was less than that of TxB₂ formation. A small inhibitory effect of TPCK on the release of 20:4 from platelet phospholipids was also observed. This evidence indicated that while a number of reactions are inhibited by TPCK, the primary effect appears to be inhibition of thromboxane synthetase.     

Regulation of microvascular prostacyclin and thromboxane with inhibitors or arachidonic acid metabolism

The levels of the stable degradation products of prostacyclin (PGI₂) and thromboxane A₂ (TXA₂): 6-oxo-prostaglandin F_1α(6-oxo-PGE_1α) and thromboxane B₂ (TXB₂) respectively were determined in the effluent of the rabbit epigastric skin flap after infusion of exogenous arachidonic acid. The blood to the flap passes through the microcirculation and thus the changes in eicosanoid biosynthesis in this part of the vasculature were recorded. The aim was to use inhibitors of arachidonic acid metabolism to increase the PGI₂/TXA₂ ratio. This may be potentially beneficial to ischaemic skin flaps by reducing platelet aggregation associated with damaged microvascular endothelium, overcoming vasospasm and increasing microvascular blood flow. Increased PGI₂/TXA₂ ratios (up to 5-fold) were best achieved using TXA₂ synthetase inhibitors such as dazoxiben hydrochloride. These were significantly more potent than the phosphodiesterase inhibitor dipyridamole, and the lipoxygenase inhibitor Bay g6575. No increase in blood flow was achieved. The cyclooxygenase inhibitor indomethacin did slow the blood flow at high concentrations (above 10⁻⁵ M), and inhibited both PGI₂ and TXA₂ synthesis. Approximately 2-fold higher concentrations of dazoxiben hydrochloride and dipyridamole were required to produce the same TXA₂ synthetase inhibition in the flap microvasculature compared with platelets .     

Improved high-performance liquid chromatographic method for the combined analysis of phospholipase, lipoxygenase and cyclooxygenase activities

We report herein an improved method for the high-performance liquid chromatographic separation and analysis of eicosanoids formed during the stimulation of human platelets in vitro with collagen. Since the products of interest, excepting arachidonic acid, contain hydroxyl groups (one to several), our method involves the conversion of the hydroxyl groups into acetates (pyridine/acetic anhydride) after derivatization with anthryl diazomethane (ADAM) rendering the compounds with much decreased polarity for separation on a reversed-phase column. This procedure is superior to that involving ADAM esters only, i.e. with free hydroxyl groups, as it leads to the excellent separation of the desired compounds from each other and from extraneous peaks observed due to the ADAM reagent and sharpens the peak of thromboxane. We have successfully applied the method to investigate the formation of thromboxane B₂ and 12-hydroxyheptadecatrienoic acid (HHT) (products of cyclooxygenase and thromboxane A₂ synthase), 12-hydroxyeicosatetraenoic acid (12-HETE, a 12-lipoxygenase product) and arachidonic acid (AA, product of phospholipase A₂) formed during the in vitro aggregation of human platelets induced by collagen. A correlation between the inhibition of aggregation by aspirin and thromboxane/HHT formation was observed. All four compounds can be chromatographed in a single run. We employed prostaglandin B₁ (PGB₁) as internal reference standard to quantify the products. The method is useful to investigate selectivity of drugs which may affect either or all of these enzyme pathways at the same time.     

Sex differences in platelet thromboxane and arterial prostacyclin production in control and n-6 fatty acid supplemented rats

Sex differences in eicosanoid production in platelets and vessel walls have been studied in control and n-6 fatty acid supplemented rats. In platelet rich plasma (PRP) of control female rats, arachidonic acid (AA) levels in phospholipids (PL), thromboxane B₂ (TxB₂) formation following collagen stimulation and aggregatory responses to collagen were higher than in PRP of male rats. 6 keto PGF_1α release from PRP-perfused isolated aortas were the same for both sexes, but the antiaggregatory activity of the wall was higher in males than in females, in association with a greater sensitivity of male platelets to prostacyclin.The administration of n-6 fatty acid supplements increased AA level in PL, TxB₂ production and aggregation only in male platelets. Production of 6 keto PGF_1α and the antiaggregatory activity of aortic walls were reduced after dietary treatment in males, but biochemical and functional parameters were not correlated in females.The results indicate complex sex-related differences in fatty acid metabolism and eicosanoid production, and in responses to n-6 dietary fatty acids in platelets and the vascular system in the rat.